Dynamic nucleolar phase separation influenced by non-canonical function of LIN28A instructs pluripotent stem cell fate decisions

LIN28A is important in somatic reprogramming and pluripotency regulation. Although previous studies addressed that LIN28A can repress let-7 microRNA maturation in the cytoplasm, few focused on its role within the nucleus. Here, we show that the nucleolus-localized LIN28A protein undergoes liquid-liquid phase separation (LLPS) in mouse embryonic stem cells (mESCs) and in vitro. The RNA binding domains (RBD) and intrinsically disordered regions (IDR) of LIN28A contribute to LIN28A and the other nucleolar proteins’ phase-separated condensate establishment. S120A, S200A and R192G mutations in the IDR result in subcellular mislocalization of LIN28A and abnormal nucleolar phase separation. Moreover, we find that the naive-to-primed pluripotency state conversion and the reprogramming are associated with dynamic nucleolar remodeling, which depends on LIN28A’s phase separation capacity, because the LIN28A IDR point mutations abolish its role in regulating nucleolus and in these cell fate decision processes, and an exogenous IDR rescues it. These findings shed light on the nucleolar function in pluripotent stem cell states and on a non-canonical RNA-independent role of LIN28A in phase separation and cell fate decisions.

e.It is unclear if LIN28A leads to nucleolus formafion, or is simply incorporated into the nucleolus.The authors seem to suggest that LIN28A and rRNA phase separafion is sufficient to form the nucleolus (from the fitle of the paper and the in vitro data).But most likely LIN28A just incorporates into the exisfing nucleolus.If that's the case, then Figure 2 will not make much sense since deplefing rRNA will only affect LIN28A phase separafion indirectly by disrupfing the nucleolus.The authors need to be less ambiguous about this important aspect of the paper.
2. A lot of essenfial data is also descripfive/only showing representafive images, lacking quanfificafions This is one of the main concerns of the paper.For example, the authors didn't use quanfitafive methods to show their data, but instead used descripfive and ambiguous ways of describing the nucleolar morphology (for example, using terms such as "floc", "diffused nucleoli", "fragmented nucleoli", "wreath structure").These terms for a non-expert reader are hard to understand, and can potenfially introduce biases when they quanfify their images.Figures such as 2g, 3c, 4e, and 5h etc will benefit greatly from quanfificafions.
3. The arficle seems to compile a number of experiments without clear explanafion of their results.The arficle as a whole has a lot of results that are not discussed very well, or seems out of place.For example, the cold-responsiveness of LIN28A phase separafion seems out of place.I am also not sure what it means to have a slower FRAP recovery after hex treatment.The authors need to connect and discuss their data instead of simply presenfing them.
Other points: 1. Lines 104-105: data in Fig. 2f doesn't support the claim that LIN28A expression level doesn't change: there is a decrease in LIN28A at 42C.GAPDH blot also seems to be cropped/manipulated.
2. Fig. 1i: separafion of LIN28A from FBL doesn't automafically mean it colocalizes with other markers of GC and DFC.To demonstrate this, the authors need to show colocalizafion of LIN28A with GC and DFC.
3. Line 128: in vivo should be changed to in cell.
4. Line 199: the authors didn't include the in vitro data showing poor solubility and prone to aggregafion.

Figure 5g, y-axis should be "relafive irregularity of FBL"
In this study, Tan and colleagues investigated the role of LIN28A and nucleolar phase separation in pluripotent stem cells.The authors show that LIN28A undergoes phase separation in the nucleolus of pluripotent stem cells.They also demonstrate rRNA is essential for the phase separation of LIN28A and associated nucleolar proteins, such as FBL and NCLs.Furthermore, they show both LIN28A IDR and RBD domains are critical for the phase separation of LIN28A and associated nucleolar protein, and also find the key amino acids critical for the phase separation.Finally, using Lin28a KO cells and these Mut LIN28A, they suggest that the non-canonical phase separation property of LIN28A regulates nucleolar remodeling and cell fate decisions of pluripotent stem cells.
We appreciate the reviewer's brief and comprehensive summary and thank for the helpful comment on our findings.Here we provide our full point-by-point responses below.
Major concerns 1.It is important to clarify the difference of the phase separation and other functions of LIN28A.Thus, it is required to demonstrate the only affecting phase separation but not other functions of LIN28A has resulted in the affects.More data need to be provided.RESPONSE: We thank the reviewer for this constructive comment.LIN28A was originally discovered as a heterochronic gene regulating developmental timing in worms 1 .Conventionally, the role of LIN28A in cell fate decision is mainly attributed to its cytoplasmic functions in binding the precursors of microRNA let-7 and certain mRNA [2][3][4][5] .Mouse Lin28A has two folded RNA binding regions, the CSD and the CCHCx2, connected by a flexible linker of 14 amino acids, with extensions of ~30 residues at both the amino and carboxyl termini.
In this previous paper (PMID: 22078496), the authors showed that the CSD provides a larger contact and contributes to let-7 affinity, but the latter domain has additional effector functions, and neither the terminal nor linker regions outside of the folded domains (CSD and CCHCx2) are essential for blocking let-7 in vivo 6 .
In mice and human, the let-7 family miRNAs comprise 12 members, the mature miRNA sequence of which is highly conserved between the different genes.LIN28A binds precursor forms of let-7 miRNAs and can inhibit both pri-let7 processing by Drosha and pre-let7 processing by Dicer 6 -Fb cfXYf hc UXXfYgg h\Y uch\Yf ZibWh]cbgv giW\ Ug NJ> V]bX]b[ fU]gYX Vm the reviewer, we first focused our analysis on the pre-let-7g, as previous studies had indicated its importance in direct association with LIN28A.
Mouse recombinant LIN28A was prepared as described in the main text of the revised manuscript.Using electrophoretic mobility shift assay (EMSA), we analyzed a series of single amino acid mutants of LIN28A to distinguish whether these mutants affected the phase separation function or the let-7 binding function of LIN28A.Both the S120 (Located in the linker disordered region) and R192 (located in the C terminal disordered region), S200 (Located in C-terminal disordered regions) mutations did not affect the LIN28A binding to pre-let-7g, but the mutation of F47 (Located at the CSD domain) abolished LIN28A binding to pre-let-7 (Response Figure 1a,1c).F47 has already been identified as a single amino acid residues required for binding to pre-let-7g, so it is used as a positive control for pre-let-7 binding.In agreement with the EMSA result, qPCR analysis also showed that S120, S200 and R192 can be mutated without affecting the expression of mature let-7g(Response Figure 1b).
As a RNA-binding protein, LIN28A can also bind mRNA (such as Ndufb10 RNA, an OxPhos Gene mRNA) and regulates stem cell metabolism 5,7,8 .Using electrophoretic mobility shift assay (EMSA), we found that WT LIN28A and LIN28A IDR point mutations had similar capacity for binding to Ndufb10 mRNA(Response Figure 1d).
Together the above data demonstrated that the IDR mutations did not affect HFJ17>xg function in RNA binding and regulation, at least for the microRNA let-7g and the mRNA we tested.
In terms of phase separation, the localization and morphology of these mutant LIN28A protein in the nucleolus were changed compared with WT LIN28A, but the localization and morphology in the cytoplasm was unchanged(Response Figure 1e).FRAP analysis also showed that these mutant LIN28A protein had lower fluidity in the nucleolus, and so as the FBL protein(Response Figure 1f,1g).To ultimately confirm the nucleolar disruptive role of LIN28A mutants was resulted from their weakened phase separation property, we generated the rescuing LIN28A mutants by fusing the exogenous IDR of FUS (S120A-FUS IDR, S200A-FUS IDR, R192G-FUS IDR), which is known to drive phase separation.Notably, the fused IDRs rescued the morphology and phase separation capability of the LIN28A mutants(Response Figure 1e,1f).Meanwhile, the three IDR fusions completely rescued the impaired fluidity of FBL caused by the LIN28A mutants(Response Figure 1g).
Together, these results demonstrated that the key amino acids at the IDR regions were essential for LIN28A phase separation function in the nucleolus, as well as for maintaining normal nucleolar integrity.On the other hand, these IDR mutations did not affect HFJ17>xg canonical functions such as RNA binding and regulation, at least for the microRNA let-7g and the mRNA we tested.We can not completely rule out there are other RNAs that can be affected by these IDR mutations, nor rule out other functions beyond RNA binding.But what is certain is that these mutations do affect LIN28A nucleolar phase separation and nucleolar integrity.

RESPONSE:
We thank the reviewer for this comment.The mouse and human naive vs primed regulation mechanisms are different.LIN28A itself does not play a role in the human naive-to-primed transition, as discussed below, therefore, it is not necessary to study its jUf]Ubhgx function in the context of naive-to-primed transition.However, according to the fYj]YkYfxg comment about mouse and human iPSCs, now we have further performed reprogramming of mouse embryonic fibroblast cells into mouse iPSCs in the revised manuscript (in addition to the reprogramming of human fibroblast cells into human iPSCs in the original manuscript).LIN28A IDR point mutations led to reduced reprogramming efficiency in this new mouse reprogramming experiment.
Moreover, to further validate the inhibitory role of LIN28A mutants on reprogramming resulted from their impaired phase-separation capacity, we generated the rescue LIN28A variants by fusing the IDR of FUS, which is known to drive phase separation.FUS IDR fusion rescued the the reduced reprogramming efficiency (Response Figure 2a,2b).
We further characterized the mouse iPSCs.Stimulated Emission Depletion Microscopy (STED) imaging revealed significant differences in the morphology and stratification of the nucleoli between MEF cells and mouse iPSCs.In the MEF cells, NPM was in irregular shape, whereas in the iPSCs+ JLI g\ckYX h\Y fcibX w`chig fcchx ghfiWhifY UbX kUg colocalized with LIN28A(Response Figure 2c).We further quantified the regularity of the granular component (GC) using Boyce-Clark semidiameter index which was originally used to assess h\Y wWcadUWhbYggx cZ gdUWY `Umcihg 9 .GC showed higher degree of regularity in iPSCs compared with MEF(Response Figure 2e).Besides, the STED imaging showed the DFC WcadcbYbh C?H hYbXYX hc g\ck h\Y wf]b[x ghfiWhifY ]bX]WUh]b[ acfY XYjY`cdYX AC@ ib]hg UbX was embedded and immersed within the granular component NPM in the iPS cells(Response Figure 2d,2f).This demonstrated the nucleoli were clearly stratified(Response Figure 2g).OP-Puromycin staining indicated that iPSCs possessed higher protein synthesis rate compared with MEF cells, suggesting the more clearly stratified nucleoli in iPSCs were functionally more developed (Response Figure 2h).
Taken together, these results demonstrated that in the context of mouse, the IDR region of LIN28A that regulated phase separation played an important role in reprogramming and mouse iPSCs.
Finally, the situation and the regulation mechanisms for mouse and human naive and primed states are different, LIN28A does not have a reported role in human naive-to-primed state transition and is not a marker of naive or primed state [10][11][12][13] .Our qPCR analysis was also consistent with this conclusion: unlike other naive (KLF4, KLF17, DPPA3) and primed (CD24) genes that were down-regulated or up-regulated respectively during human naive-toprimed conversion, LIN28A did not change its expression level during this process (Response Figure 2i,2j).3. The authors conclude that the nucleolar phase separation property, but not the canonical microRNA let-7 binding properties of LIN28A, that could mediate the naive-to-primed state conversion.It seems that a considerable portion of these mutant LIN28A protein is located in the cytoplasm, which may bind to RNA target.The authors need to provide data evidences that these mutant LIN28A lose its binding to microRNA let-7.

RESPONSE:
We thank the reviewer for this good and constructive comment.In the process of our experiments, WT Lin28A and the three LIN28A IDR mutants had similar localization and fluidity in the cytoplasm.Using electrophoretic mobility shift assay (EMSA), we found that WT Lin28A and LIN28A IDR point mutations had similar capacity for binding to microRNA let-7(pre-let-7g)(Response Figure 3a).qPCR analysis also showed that S120, S200 and R192 can be mutated without affecting the expression of mature let-7g (Response Fig. 3b).These data illustrated that the IDR mutations did not affect LIN28A binding to microRNA let-7, nor let-7 expression level.
On the other hand, to prove that the IDR mutations can cause disruption of nucleolar phase separation through the phase separation function of LIN28A, we generated the rescuing LIN28A mutants by fusing the exogenous IDR of FUS (S120A-FUS IDR, S200A-FUS IDR, R192G-FUS IDR), which is known to drive phase separation [14][15][16] .Notably, the fused IDRs rescued the morphology and phase separation capability of LIN28A itself and other nucleolar protein FBL in the LIN28A mutants(Response Figure 3c, 3d, 3e). 4. The authors show that phosphor-null LIN28A lose phase separation property and capacity to promote naïve-primed transition and reprogramming.They also propose that LIN28A is important for the phase stratification of nucleoli during reprogramming.Is phosphorylation of LIN28A important for the function of LIN28A in pluripotent stem cell fate?The authors needs to measure LIN28A phosphorylation in naïve-primed transition and reprogramming, and provide data of LIN28A phosphorylation function in naïve-primed transition and reprogramming.

RESPONSE:
We thank the reviewer for this helpful suggestion.In this previous paper (PMID: 27992407)(Response Figure 4a), the authors have demonstrated that LIN28A S200 phosphorylation contributes to the regulation of reprogramming or pluripotency state transition 17 .
First, it showed that the phosphor-null (S200A) and phospho-mimetic (S200D) LIN28A led to approximately 50% decreased or increased reprogramming efficiency(Response Figure 4b), respectively, indicating that S200 phosphorylation had an important role in the induction of pluripotency.
They then explored the regulation of LIN28A levels in mESCs cultured in serum/LIF versus 2i/LIF naive state conditions.Total LIN28A protein and phosphorylated LIN28A levels were reduced in the 2i/LIF culture(Response Figure 4c).In the serum/LIF condition (or a metastable state close to the primed state), due to the presence of the ERK signaling, the S200 of LIN28A is phosphorylated which leads to higher stability of the LIN28A protein(Response Figure 4d,4e,4g).So in the primed state or the serum/LIF condition, the total LIN28A level, as well as the phosphorylated LIN28A level are higher compared with the naive state.On the other hand, in the naive condition, because the ERK signaling is inhibited, S200 of LIN28A is not phosphorylated(Response Figure 4f), and thus LIN28A protein stability is lower, and the protein level is also lower.This is how the phosphorylation of S200 of LIN28A regulates the naive/primed pluripotent states.
They then performed clonogenic assays upon transfer from 2i/LIF to serum/LIF and assessed the alkaline phosphatase (AP) staining pattern of colonies emerging in the serum/LIF culture, which is characterized by a mix of compact, uniformly AP-positive, naïve-like 'ugc`]Xv( colonies and larger, heterogeneously AP-stained, more primed 'ua]lYXv( colonies.The S200D phospho-mimetic mutant showed a reproducibly higher number of solid colonies relative to wild-type LIN28A(Response Figure 4h), demonstrating that the higher LIN28A protein level mediated by S200 phosphorylation enhances LIN28>xg function in promoting the transition from naïve to primed pluripotency.
Together, these reprogramming and mESC data demonstrate that LIN28A phosphorylation contributes to reprogramming and the regulation of pluripotency state transition.[REDACTED] 5. What is the function of the phase separation of LIN28A in differentiation of pluripotent stem cells.The authors need to provide data of mouse and human cells.RESPONSE:We thank the reviewer for this good suggestion.The undifferentiated state of mouse ES cells is maintained in the presence of leukemia inhibitory factor(LIF) in the culture medium [18][19][20] .The spontaneous differentiation of ES cells can be triggered by withdrawal of LIF from the medium 21,22 .
In our experiments, the spontaneous differentiation was induced by the withdrawal of LIF 12 h after plating E14 ESCs.Cell pellets were collected at 3, 5, and 7 days after LIF withdrawal and induction of differentiation, and then qPCR analysis showed that the expression of Lin28a decreased sharply after LIF withdrawal in 3 days (Response Figure 5a).The IDR mutated LIN28A variants can slightly delay the differentiation of pluripotent stem cells (Response Figure 5b).The possible reason is that these mutants had the tendency to stay in a state closer to the naive state, and had slower kinetics to exit the pluripotency as illustrated in the Response Figure 3g, 3f, 3h above.As LIN28A does not play a role in human naive pluripotency exit, as illustrated in the Response Figure 2i, 2j, it is not necessary to examine the human situation, and we use the mouse ESC exit from pluripotency and induction of differentiation here to address the question.
Besides, it is well-established that LIN28A is a pluripotency factor.LIN28A was abundantly expressed in undifferentiated ESCs, embryonal carcinoma cells and early embryonic tissue, but declines in expression and becomes tissue restricted.Because LIN28A is decreased during differentiation, we expect that it does not have a role in regulating differentiation genes.Our qPCR analysis also showed that the IDR mutant LIN28A did not affect the expression kinetics of genes in the three germ layers(Response Figure5c,5d,5e).
Response 6.Besides RNA, other proteins, ions or components are involved in the phase separation of LIN28A?In vitro and in vivo mouse and human data should be provided.

RESPONSE:
We thank the reviewer for bringing our attention on this issue.The LIN28A protein and RNA were mixed at the indicated concentrations.Then in vitro phase separation assay was performed in a reaction buffer containing 50mM HEPES, 50mM NaCl, 1mM DTT and 10% PEG.
Fhwg worth noting that low salt promotes LIN28A phase separation in vitro.At first, we tried to observe phase separation in a reaction buffer containing 50mM HEPES, 150mM NaCl (physiological salt conditions), 1mM DTT and 10% PEG, the solution of LIN28A proteins remained clear at room temperature(by visual inspection), and when examined by light microscopy, only the irregular aggregation were observed(Response Figure 6a#6b).
However, when the NaCl concentration was diluted to 50mM, solutions of LIN28A proteins became opalescent(by visual inspection), and round structures were observed by light microscopy(Response Figure 6a,6b).
The temprature plays a role in phase separation.LIN28A was localized in both the nucleus and cytoplasm at 37°C (Response Figure 6c).When cells were exposed to cold shock (25°C), LIN28A tended to be reduced in the nucleus (Response Figure 6c).When cells were exposed to heat shock (42°C) for 15min, LIN28A tended to become more compact compared with that at 37°C (Response Figure 6c).Next, we investigated the difference in fluidity of the LIN28A phase-separated condensate at 37°C, 25°C and 42°C.Fluorescence recovery after photobleaching (FRAP) analysis of LIN28A-eGFP was performed at the indicated temperatures.After bleaching for 300 seconds, fluorescence signals of LIN28A in the nucleus had 20-30% of recovery in all the three conditions, with no significant difference in the degree of LIN28A recovery (Response Figure 6d).In contrast, the fluidity of LIN28A in the cytoplasm had above 40% recovery and the 42°C condition had over 60% recovery (Response Figure 6d).Statistical analysis showed that cold shock decreased LIN28A loci numbers in the nucleus and the nucleus : cytoplasm intensity ratio, whereas heat shock increased both (Response Figure 6e,6f).We also observed more dispersed areas of LIN28A in cold-shocked nuclei and more compact areas of LIN28A in heat-shocked nuclei (Response Figure 6g).These results suggested that cold shock promoted LIN28A outflow from the nucleus and the condensate became more diffused, while heat shock promoted its inflow into the nucleus and the condensate became more compact.
The intrinsically disordered regions (IDRs) obviously play a role.A hallmark for LLPS assemblies for many proteins, is the presence of IDRs.These regions are enriched in repetitive sequences of a few amino acids, usually resulting in characteristic domains of low complexity 23 .Besides RNA, the N/C-terminal and Linker IDRs promoted the establishment of the LIN28A protein phase separated condensate both in vitro(Response Figure 6i,6j) and in vivo(Response Figure 6h).The LIN28A truncation experiment and IDR mutation experiment demonstrated that the IDR regions were essential for LIN28A phase separation.
Regarding other proteins, we mainly investigated them in vivo, such as FBL and NCL, but not in vitro, because the in vitro reconstituted recombinant proteins can't actually reflect the real situation in vivo.
Regarding human and mouse LIN28A recombinant proteins, there are 97% similar in their protein sequence(Response Figure 6k), and mouse LIN28A protein is frequently used to indicate functions of LIN28A of both mouse and human 6 .In this paper by Tan et al., the authors reported that LIN28A, an RNA binding protein could le[\i^f elZc\fcXi g_Xj\ j\gXiXk`fe+ n_`Z_ nXj c`eb\[ kf IFK17>zj XY`c`k`\j kf `e[lZ\ eXrm\ kf primed state transition in mouse embryonic stem cells.The authors pinpointed the domains and residues in LIN28A that assisted their phase separation.These domains also happened to be important for naïve to primed pluripotency transition.The paper is potentially interesting to the fields of phase separation, nucleolar biology, and stem cell differentiation.However, it suffers from technical deficiencies in phase separation characterizations.A lot of essential data is also descriptive/only showing representative images, lacking quantifications.The article seems to compile a number of experiments without clear explanation of their results.Therefore, my enthusiasm for the paper is dampened.
Major concerns: 1. Technical challenges in phase separation experiments: a.Most of the phase separation experiments are done using an over-expressed LIN28A-GFP construct.How its behavior compares to endogenous LIN28A localization/behavior is unknown.The STORM image showing endogenous LIN28A organization (Fig. 1i) looks different from overexpressed LIN28A-GFP.RESPONSE: We thank the reviewer for this constructive suggestion.Following this guidance, we have generated the eGFP-LIN28A knock-in mESCs line(Response Figure 7a,7b).LIN28A had the similar localization in the nucleolus and cytoplasm in the wild-type ES cells (by immunofluorescence staining), eGFP-LIN28A-overexpressed (live imaging), and eGFP-LIN28A knock-in mESCs line (live imaging) (Response Figure 7c) .
Next, we tested the fluidity of LIN28A protein condensates both in the nucleolus and cytoplasm, FRAP experiments indicated that overexpressed LIN28A exhibited similar fluidity with the knock-in LIN28A(Response Figure 7d).
Due to the differences in image processing, the STORM image looks like a cartoon(we removed in our revised manuscript).In fact, LIN28A immunostaining in the wild-type, overexpressed eGFP-LIN28A and eGFP knock-in LIN28A live imaging all showed that LIN28A in the nucleolus formed a round shell with holes, and FBL was embedded in the holes.LIN28A was diffusely distributed in the cytoplasm (Response Figure 7c).
Response Figure 7 WT Knockin If they bleached the whole, since there is not much LIN28A in the nucleoplasm, it is understandable that the recovery will be slow and not complete, because there is simply not much LIN28A surrounding the nucleolus to diffuse from.If this methodology is not established, any difference they see comparing WT and mutant LIN28A does not make much sense.It is also unclear FRAP in the cytoplasm is done.Are there cytoplasmic condensates of LIN28A too?RESPONSE: We thank the reviewer for bringing our attention to this issue.For the FRAP experiments, we bleached half of the nucleolus or a portion of the nucleolus and showed the bleach images as below(Response Figure 9a-9l).Therefore, we believe we have employed the right methodology to evaluate the fluidity of nucleolus, because with our bleaching method the LIN28A protein can diffuse from the area outside of the bleached area, as the reviewer suggested.
In our experiment, we can see that LIN28A was generally diffusely distributed in the cytoplasm.We ZXezk Zfdgc\k\cp \oZcl[\ k_\i\ Xi\ jfd\ ^iXelc\j ]fid\[ `e k_\ ZpkfgcXjd+ such as the stress granules in certain stress conditions.FRAP experiments showed that LIN28A in the cytoplasm exhibited higher recovery compared to that in the nucleolus.Also WT type LIN28A and those LIN28A mutations showed different fluidity in the nucleolus, but had similar fluidity in the cytoplasm(Response Figure 9g,9h).Based on these reasons, we found it is more interesting to study the phase separated LIN28A in the nucleolus, therefore we focused on the nucleolar LIN28A condensates in this study.After-bleach l d.The authors found most of the domains of LIN28A are important for proper organization of nucleolus.However, they decided to say that only IDRs are important, and are the ones they follow up with using individual mutations.This is a bit puzzling and needs better explanation.

RESPONSE:
We thank the reviewer for bringing our attention to this issue.LIN28A contains two well-known RNA-binding domains(RBDs), a cold-shock domains (CSD), and a cysteine cysteine histidine cysteine (CCHC) zinc-finger domains (ZFD).A flurry of studies y showing that LIN28A performedhas an important role in reprogramming and maintenance of pluripotency roles through let-7 dependent(binding to let-7) or independent (binding directly to mature mRNA) pathways based on its RNA-binding domains 4,6,24 .
We made the truncated mutants of RBDs and IDRs (Figure3), and found that both RBDs and IDRs of LIN28A were important for proper organization of nucleolus.The RNA-binding domains have been studied intensively in the context of LIN28 function.We were more curious about the function of IDRs, which was previously assumed to have no functional roles in LIN28Ain a protein is frequently assumed to be diagnostic of its ability to phase separate.Therefore, iIn this article, we focus on IDRs that have not been rarely intensively studied before.
To clarify this rationale for choosing IDR as the focus of this study, we have added more discussion in the Discussion section of the revised manuscript.
e.It is unclear if LIN28A leads to nucleolus formation, or is simply incorporated into the nucleolus.The authors seem to suggest that LIN28A and rRNA phase separation is sufficient to form the nucleolus (from the title of the paper and the in vitro data).But most likely IFK17> aljk `eZfigfiXk\j `ekf k_\ \o`jk`e^elZc\fclj-F] k_Xkzj k_\ ZXj\+ k_\e C`^li\ 1 n`cc efk make much sense since depleting rRNA will only affect LIN28A phase separation indirectly by disrupting the nucleolus.The authors need to be less ambiguous about this important aspect of the paper.

RESPONSE:
We thank the reviewer for bringing our attention to this issue.In terms of our current results, we should tune down our conclusion.LIN28A is indeed integrated into nucleolus.LIN28A wraps around GC(NPM) in the nucleolus )(Figure 1c,6c in the revised manuscript).The cavities in the LIN28A inclusions tended to encapsulate DFC(FBL)(Figure 1b,1c in the revised manuscript).Loss of LIN28A resulted in disrupted stratification of nucleoli, but the disordered nucleoli were still present(Figure 5e in the revised manuscript).These results demonstrated LIN28A itself is not sufficient to form nucleolus, but its loss impairs the integrity of nucleolus.We can consider LIN28A as a marker of nucleolar integrity, it acts as a solid shell or scaffold to help stabilizing the existing nucleolar layered structure.
In Figure 2, we just used LIN28A as a marker of nucleolar integrity, and `kzj highly possible that disrupting rRNA indirectly affected LIN28A through first disrupting the nucleolar integrity.We also tune down the title from yApeXd`Z Nucleolar Phase Separation  3. The article seems to compile a number of experiments without clear explanation of their results.The article as a whole has a lot of results that are not discussed very well, or seems out of place.For example, the cold-responsiveness of LIN28A phase separation seems out of place.I am also not sure what it means to have a slower FRAP recovery after hex treatment.
The authors need to connect and discuss their data instead of simply presenting them.

RESPONSE:
We thank the reviewer for bringing our attention on this issue.We agree with this suggestion that the cold-responsiveness of LIN28A phase separation seems out of place.
We have removed it in the revised manuscript. .
Mammalian nucleolus was a multiphase liquid condensate.The aliphatic alcohol 1,6hexanediol(HEX) interferes with weak hydrophobic interactions and is often used to dissolve protein condensates in cells 23 .LIN28A is present both in the nucleolus and cytoplasm.We can use HEX to study in which compartment can LIN28A form condensate. Confocal imagings and the FRAP experiment indicated that HEX treatment did not affect cytoplasmic LIN28A distribution, suggesting that cytoplasmic LIN28A was more diffused and did not have typical phase-separated condensate behavior.However, the nucleolar LIN28A was disrupted and the fluidity slowed considerably after HEX treatment, suggesting that nucleolar LIN28A assumed the phase-separated condensate features.
We have added this rationale in the revised manuscript: Line93-1079 wJXddXc`Xe nucleolus was a multiphase liquid condensate.The aliphatic alcohol 1,6-hexanediol(HEX) interferes with weak hydrophobic interactions and is often used to dissolve protein condensates in cells.LIN28A is present both in the nucleolus and cytoplasm.Thus we used HEX to study in which compartment can LIN28A form condensate. Endogenous eGFP-LIN28A knock-in ESCs treated with 1% HEX for 10 minutes showed diffusion of the condensates in the nucleolus (Fig. 1d).To test the fluidity of LIN28A protein condensates, we performed FRAP experiments and found that LIN28A in the nucleolus exhibited slower recovery compared to that in the cytoplasm, and the nucleolar LIN28A condensate was more sensitive to the HEX treatment (Fig. 1e and Extended Data Fig. 5a).The statistical analysis also quantitatively showed a reduction in LIN28A condensates intensity in the nucleolus after HEX treatment (Fig. 1f), and an increase in the dispersed area and irregularity of LIN28A in the nucleolus after HEX treatment (Fig. 1g,h).HEX treatment did not affect cytoplasmic LIN28A distribution, suggesting that cytoplasmic LIN28A was more diffused and did not have typical phase-separated condensate behavior.In contrast, the nucleolar LIN28A was disrupted and the fluidity slowed considerably after HEX treatment, suggesting that nucleolar LIN28A assumed the phase-separated condensate ]\Xkli\j-x The authors did experiments to answer some quesfions.Quesfions are remaining to be answered.And the data should be clear explanted and connected to be form a story.
1，It is important to clarify the difference of the phase separafion and other funcfions of LIN28A.In the revision, the authors only test the microRNA let-7g and one mRNA in the IDR mutafion.This can not rule out other RNAs that can be affected by these IDR mutafions.Also the authors conclude that phosphornull LIN28A lose phase separafion property and capacity of cell fate.Here the difference of the phase separafion and other funcfions of LIN28A also need to be clarified.The whole genome-wide Seq is suggested to invesfigate the RNA binding and regulafion and sequenfial funcfions.
2, It is sfill unclear whether and how cytoplasmic condensates of LIN28A, and the interacfion and molecular linking mechanism between nucleolar and cytoplasmic condensates of LIN28A.Suggest more data to elucidate the cytoplasmic and nucleolar condensates of LIN28A, which one loses the funcfions of RNA binding, and funcfion of mouse/human iPSC and mouse naïve-to-primed funcfion.The colocalizafion of LIN28A is puzzling.The results in the revision is controversial from the original data.The solufion of image is not the level of supersolufion of STED, but as the normal fluorescent microscopy.3, The funcfion of the phase separafion of LIN28A in human pluripotent stem cell differenfiafion is sfill lacking.And instead of carefully invesfigafing the temperature regulafing LIN28A, the authors directly delete this part.Suggest further invesfigafion and data to clearly show the hot and cold condifion on LIN28A expression and phase separafion.
4,It is interesfing to find that both RBDs and IDRs of LIN28A were important for proper organizafion of nucleolus.However, the authors only focused on IDRs, which is well known important for phase separafion, and ignore RBDs.Suggest mutafions of RBDs in overexpression and knock-in system to invesfigate its role.And the mechanism of LIN28A leading to nucleolus formafion or simply being incorporated into the nucleolus is sfill unclear.The relafionship between LIN28A, rRNA and construcfive nucleolus protein machine needs to be invesfigated.Besides in vivo system, the in vitro nucleosome assembly structure by EM and physical quanfity is suggested to answer this.

Minor:
The added data such as Fig 2h,3d,4ghi, 6f,S2c S4d, lacking stafisfics.The western and also other data also lacking stafisfics.Three or more independent biological repeats should be required.

Reviewer #2 (Remarks to the Author):
In this revised manuscript, the authors included a lot of new data, including the LIN28 CRISPR KI cell line and image data quanfificafions.I have a few more comments before the manuscript can be accepted: 1) There is sfill confusion here: "However, the nucleolar LIN28A was disrupted and the fluidity slowed considerably after HEX treatment, suggesfing that nucleolar LIN28A assumed the phase-separated condensate features."Do the authors mean that LIN28A only assumed condensate features before or after Hex treatment?The authors need to be a bit clearer in their wrifing.I also don't get the logic that slower FRAP recovery after Hex shows it is phase separated.There is no basis for claiming this.Usually Hex is just used to dissolve the condensates.I would suggest to just delete the Hex data if it is not relevant elsewhere.
2) There are more "in vivo" in the text that needs to be changed to "in cell"."In vivo" is usually used to describe in an animal.
efficiency of MEF cells transduced with OCT4, SOX2, NANOG, and LIN28A variants.(also new Fig.6a in the revised manuscript) b.Number of iPS colonies 14 days after OSNA transduction of MEF cells.n = 3 biologically independent experiments, error bar: standard error of the mean,*p < 0.05, **p <0.01,***p < 0.001; One-way ANOVA.(also new Fig.6b in the revised manuscript) c.Representative STED immunofluorescence images of LIN28A and NPM in MEF or iPS cells.(also new Fig.6c in the revised manuscript) d.Representative STED immunofluorescence images of NPM and FBL in MEF or iPS cells.(alsonew Fig.6din the revised manuscript)

Finally, we showed
by NANOG immunostaining(Response Figure 3f,3g) and qPCR analysis(Response Figure 3h) that the IDR mutants led to reduced naive-to-primed conversion efficiency, and it was through the phase separation function of LIN28A because the FUS IDR fusion could completely rescue the defects caused by the IDR mutants. .EMSAs with pre let-7g 7g as the probe, mixed with increasing concentrations (0.02, 0.1, 0.2, 0.5, and 1µM) of mouse LIN28A variants.(also new Extended Data Fig.8c in the revised manuscript) b. b. qRT-PCR showing the let-7g 7g expression in WT, Lin28a KO, and LIN28A mutations ESCs.n = 3 biologically independent experiments, error bar: standard error of the mean,*p < 0.05, **p <0.01,***p < 0.001,Two way ANOVA.(also new Extended Data Fig.8b in the revised manuscript) c. c.FRAP analysis showing fluorescence recovery of of WT WT-LIN28 28A, A, S120A, A, S200A, A, R192G, G, and FUS-IDR fused LIN28 28A variants after photobleaching in in the nucleus (n (n = 3) in in biologically independent experiments Data are presented as as mean values *.• SEM SEM standard error of of the mean (also Fig 4d in in the revised manuscript d. d.FRAP analysis showing FBL fluorescence recovery after photobleaching in in WT, Lin28 28a KO, and Lin28 28a KO KO cells transduced with S120A LIN28 28A , S200A LIN28 28A , R192G LIN28 28A, A, and FUS-IDR-fused LIN28 28A variants (n (n = 3) in in biologically independent experiments Data are presented as as mean values *.• SEM SEM standard error of of the mean (also Fig 4i in in the revised manuscript e. e. Representative confocal microscopy Airyscan images of of the morphology and nucleolar localization of of LIN28 28A and FBL in in living WT, Lin28 28a KO, and Lin28 28a KO KO cells transduced with Mut-LIN28 28A, A, S120A LIN28 28A, A, S200A LIN28 28A, A, R192G LIN28 28A, A, and FUS-IDR-fused LIN28 28A variants cultured in in LIF/Serum medium Scale bar, 5µm µm.(alsoFig 4f in in the revised manuscript f. f.NANOG immunostaining of WT, Lin28a KO, and Lin28a KO ESCs transduced with full length WT LIN28A, single mutation variants, or IDR-fused variants, converted from the naïve state to the primed state.Scale bar, 200µm.also Fig.5c in the revised manuscript g. g.Statistical analysis of NANOG protein fluorescence intensity of the above cells in (f).n = 64, error bar: standard error of the mean, *p < 0.05, ** **p <0.01,***p < 0.001, Two way ANOVA.(also Fig.5d in the revised manuscript h.h.qRT-PCR showing the naïve and primed pluripotent marker gene expression in WT, Lin28a KO, and Lin28a KO ESCs transduced with full length WT LIN28A or the indicated LIN28A variants, cultured in the naïve and primed state conditions.n = 3 biologically independent experiments, error bar: standard error of the mean,*p < 0.05, ** **p <0.01,***p < 0.001, Two way ANOVA.(also Extended Data Fig.7c in the revised manuscript) referenced article.Response Figure 4 is from this article.b.TRA-1-60 staining of iPSCs from a reprogramming experiment using OSN and empty vector (EV), wild-type LIN28A (WT), phospho-null (S200A) LIN28A, or phosphomimetic (S200D) LIN28A (day 21 of reprogramming).Western blot analysis of endogenous LIN28A in v6.5 mESCs cultured in serum/LIF or 2i/LIF.c.Western blot analysis of endogenous LIN28A in v6.5 mESCs cultured in serum/LIF or 2i/LIF conditions.d.Western blot analysis of transgenic wild-type (WT), phosphomimetic (S200D), or phospho-null (S200A) FLAG-LIN28A added back in LIN28A/B KO mESCs.e. Cycloheximide chase of transgenic FLAG-LIN28A variants in HeLa (Flp-In) cells.CHX= cycloheximide (100 µg/ml).f.Western blot (left) analysis of endogenous LIN28A in v6.5 mESCs after a four-hour dropout of PD0325901 (PD) or CHIR99021 (CH).g.Western blot analysis of LIN28A (S200) phosphorylation in PA1 cells after 30min stimulation with serum (10%), fibroblast growth factor (FGF) (100 ng/ul), or epidermal growth factor (EGF) (100 ng/ul).Cells were serum-starved for 16t20 h prior to stimulation.h.Alkaline Phosphatase (AP) analysis of mESCs from panel (f) grown at clonal density upon transfer from 2i/LIF to serum/LIF.Representative images of colonies with solid and mixed staining patterns are shown on the left.Scale bar = 100 µm.Quantification of the number of solid colonies is shown on the right.n=4 independent experiments.Error bars represent s.e.m. **P<0.01(two-tailed OhiXYbhxg h-test).

6 -
representation of the eGFP-LIN28A knock-in E14 mESC line generated.(also new Fig.1a in the revised manuscript) b.PCR experiments showing that the knock-in mESCs genome contains the insertedhas an extra genome of about 700 base fragments compared with wild-type (WT) mES cells.This result indicates that the LIN28A-eGFP gene was successfully knockedin(homozygotes). (also new Extended Data Fig.1a in the revised manuscript) c.Confocal microscopy Airyscan images of the morphology and nucleolar localization of LIN28A and FBL in immunostaineding wild-type E14 mESCs, live-imageding overexpressed LIN28A-eGFP overexpressing E14 mESCs, and live imageding LIN28A-eGFP knock-in E14 mESCs.Scale bar, 5µm.(also new Fig.1b in the revised manuscript) d.FRAP analysis and images showing overexpressed eGFP-LIN28A and knock-in eGFP-LIN28A recovery after photobleaching in the nucleus (n = 3) in biologically independent \og\i`d\ekj-AXkX Xi\ gi\j\ek\[ Xj d\Xe mXcl\j *.• PBJ-PBJ9 jkXe[Xi[ \iifi f] k_\ mean.Scale bar, 5µm.(also new Extended Data Fig.1b in the revised manuscript) Hexandiol experiment is shown without any rationale.It is unclear what the authors mean by Hex treatment leads to diffusion of the condensates (clearly not the case judged by Fig.1b).Plus, the concentration of Hex is too high (10% for 10 min).The nucleus should be dissolved by then.People usually use 1-3% for 10 min.RESPONSE: We thank the reviewer for this helpful suggestion.1,6-hexanediol (HEX) is used as a LLPS inhibitor in phase separation studies23  , and we used it to indicate that LIN28A ]fidj g_Xj\ j\gXiXk\[ Zfe[\ejXk\->ZZfi[`e^kf k_\ i\m`\n\izj jl^^\jk`fe+ n\ _Xm\ lg[Xk\[ and reanalyzed the experiment in the eGFP knock-in ESCs.ESCs were treated with 1% HEX for 10 minutes and we observed that LIN28A was diffused in the nucleus (Response Figure8a, 8d).We also performed FRAP experiments and found that LIN28A in the nucleolus exhibited slower recovery compared to that in the cytoplasm(Response Figure8b,8c), and the nucleolar LIN28A condensate was more sensitive to the HEX treatment, suggesting LIN28A forms more less fluidy condensate in the nucleolus than in the cytoplasm.is unclear how the authors performed FRAP experiments.Did the authors bleach half of the nucleolus, or the whole nucleolus?It is critical to explain, and to show the bleach images since to probe interior fluidity of the nucleolus, the authors should bleach half of it and analyze its recovery.

Fig 1C , 7 ,
LIN28A is oversaturated，could not draw proper conclusion.Suggest for cytosol and nucleolar one, the real supersolufion image, 3D image and/or immuno-EM to show the clear localizafion of both overexpressed and endogenous LIN28A.

also new Fig.6e in the revised manuscript) f
. Graph showing the number of typical DFC(FBL) morphology in MEF or iPSCs.n=20 nucleoli, number of nucleoli.(also

new Fig.6f in the revised manuscript) g
. A cartoon diagram showing morphological changes of NPM and FBL in MEF or iPSCs.(also

new Fig.6g in the revised manuscript)
Mediated by Non-canonical Function of LIN28A Instructs Pluripotent Stem Cell Fate A\Z`j`fejz to yApeXd`Z Nucleolar Phase Separation Influenced by Non-canonical Function of LIN28A Instructs Pluripotent Stem Cell Fate A\Z`j`fejz.